A variety of assay systems utilize ligands, e.g., nucleic acids, immobilized on the surface of a solid support. Effective immobilization of the nucleic acids is difficult, both because a range of materials are used to form the solid supports utilized in these assays, and because individual assays have special requirements. Therefore, although a number of attachment mechanisms have been developed, none are universally acceptable and most exhibit notable deficiencies. Among other drawbacks, present methods tend to require large amounts of nucleic acids, have high background noise levels or lack versatility (Duran et al. U.S. Pat. No. 5,858,653 issued Jan. 12, 1999).
The reproducible production of solid supports containing immobilized nucleic acids can also be problematic. For example, a convenient method of attachment utilizes nucleic acids with acrylamide functional groups which can be copolymerized to polyacrylamide gel matrices by free radical polymerization. However, oxidation can affect the copolymerization process resulting in variability in the results achieved using different supports, even when prepared using the same materials. Moreover, long-term stability of supports containing immobilized ligands has been difficult to achieve, often limiting the period of use to shortly after preparation.
The present invention is based, at least in part, on the discovery of a novel and convenient method of immobilizing a ligand, e.g., a nucleic acid, on a solid support. The method utilizes a covalent bond formed between a thiol group immobilized on the solid support and an acrylamide functional group contained on the nucleic acid to immobilize the nucleic acid to the support. In a particular embodiment, the covalent bond formed is a sulfide, a thioether, bond. The solid support can contain a polymer layer.
The method and the supports it produces are advantageous in several respects. The method utilizes reagents which are both readily available and compatible with the types of analysis conducted with solid supports. Because the materials can be used in aqueous solutions, the need for special skills and sophisticated chemical apparatus are minimized. In addition, because the materials and the supports they form are quite stable, the reproducibility from support to support which has previously proved so difficult to achieve can be realized. This stability also permits the components forming the bond to be combined at different times. For example, because solid supports containing the latent thiol groups of the invention are extremely stable, they can be produced under consistent conditions for use at a later time. Prior to analysis, the latent thiol groups can be activated and contacted with the acrylamide modified nucleic acids to form a support containing immobilized nucleic acids. In a particular embodiment, the thiol groups are activated by contact with a reducing agent.
In one embodiment, the invention is directed to a method of immobilizing an affinity ligand on a solid support comprising providing a solid support comprising an immobilized thiol group, contacting the thiol group with a nucleic acid comprising an acrylamide functional group, and forming a covalent bond between the two groups, thereby immobilizing the ligand on the solid support.
In a particular embodiment, the ligand is a nucleic acid, a modified nucleic acid or a nucleic acid analog. The solid support can comprise a plurality of thiol groups. A plurality of ligands can be immobilized on the solid support. In alternate embodiments, the solid support is formed from glass, silica, ceramic, plastic or metal compounds. The solid support can comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids. The solid support can further comprise a polymer layer. In a particular embodiment, the solid support can comprise a microarray. The thiol groups can comprise disulfide groups.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on a solid support comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one acrylamide functional group, thereby immobilizing an affinity ligand on a solid support.
In a particular embodiment, the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously. In alternate embodiments, the solid support is formed from glass, ceramic, plastic and metal. The solid support can comprise two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids. The solid support can further comprises a polymer layer. The solid support can comprise a microarray.
In another aspect, the invention is directed to the product formed by the method of forming a solid support described above.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on microarray comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one xcex1,xcex2 unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support. In a particular embodiment, the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on a microarray comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one xcex1,xcex2 unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support. In a particular embodiment, the ligand is a nucleic acid, a modified nucleic acid or a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously.
The method can additionally include contacting a glass solid support with a silane compound to form a solid support having an unsaturated aliphatic surface. The silane compound can be represented by Structural Formula I: 
In Structural Formula I, X is a halogen, and R1, R2 and R3 are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, provided that at least one of R1, R2 or R3 is an alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl. The unsaturated aliphatic surface is then contacted with a polymerization solution containing free radical initiator, a disulfide bisacrylamide, and optionally containing an acrylamide to form a solid support comprising immobilized latent thiol groups. Disulfide bisacrylamides can be represented by Structural Formula IIA: 
In Structural Formula IIA, n and m are each, independently, a positive integer.
The latent thiol groups can be activated by contacting the solid support with a disulfide reducing agent. When it is desirable to have a crosslinked gel having immobilized thiol groups, the polymerization solution can additionally include alkylene bisacrylamide.
In an alternative embodiment, the unsaturated aliphatic surface is then contacted with a polymerization solution containing free radical initiator, a compound having a xcex1,xcex2-unsaturated carbonyl and a protected thiol group, and optionally containing an acrylamide to form a solid support comprising immobilized latent thiol groups. The compound having an (xcex1,xcex2-unsaturated carbonyl and a protected thiol group preferably can be represented by Structural Formulas IIB-IID: 
In Structural Formulas IIB-IID, R11 and R4 are defined as above. R14 is xe2x80x94(CH2)pxe2x80x94 or xe2x80x94(OCH2CH2)pxe2x80x94. In a preferred embodiment, R4 is xe2x80x94SR15, where in R15 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl group.
In several embodiments of the invention, it is useful to provide latent thiol groups through the use of polymerizable disulfide compounds. As indicated in Structures IIB-D, such compounds can be monofunctional or bifunctional with regard to the xcex1,xcex2 unsaturated carbonyl group. A commercially available example of a bifunctional disulfide reagent is BAC. An example of a monofunctional disulfide reagent is AEMA (Schnaar, R. L. et al., 1985, Analytical Biochemistry, 151:268-281). Additional monofunctional acrylamide disulfide derivatives can be generated by reacting BAC with the reducting agents xcex2-mercaptoethanol and thioacetic acid, as shown if FIGS. 8 and 9.
In a particular embodiment, the free radical initiator is added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support.
The method can additionally include derivatizing the solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups. In a particular embodiment, the solid support includes an amine functional group and the solid support is derivatized by contacting the solid support with a compound represented by Structural Formula III: 
In Structural Formula III, Y is a leaving group, L is a linking group, and R4 is a thiol protecting group. The derivatized solid support formed has immobilized latent thiol groups.
In a particular embodiment, Y is one of the following: 
wherein R6 and R7 are each, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.
In a particular embodiment, R4 is one of the following groups: 
wherein R8 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.
In another aspect, the invention is directed to a method of preparing a solid support having immobilized thiol groups. The method includes contacting a glass solid support with a silane compound represented by Structural Formula I to form a solid support having an unsaturated aliphatic surface. The unsaturated aliphatic surface of the solid support is then contacted with a polymerization solution containing free radical initiator, a disulfide bisacrylamide represented by Structural Formula IIA-D, and optionally containing an acrylamide to form a solid support comprising immobilized latent thiol groups. The latent thiol groups of the solid support are then contacted with a disulfide reducing agent to form a solid support having immobilized thiol groups.
In one embodiment, the solid support is doped or undoped silica, alumina, quartz or glass, and the method further comprises the steps of contacting the solid support with a compound comprising a silane group or a carboxylic acid and a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface, and contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.
The compound can be represented by the following structural formula: 
wherein X is a halogen, and R1, R2 and R3 are each, independently, a halogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group or a group having at least one (xcex1,xcex2-unsaturated carbonyl, provided that at least one of R1, R2 or R3 is a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl.
The latent thiol groups can be activated by contacting the solid support with a disulfide reducing agent. The polymerization solution can further include alkylene bisacrylamide. The free radical initiator can be added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support
The solid support can be gold, silver, copper, cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten, and alloys thereof, and the method can further comprise the steps of contacting the solid support with a compound comprising a thiol group, sulfide or disulfide group and a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface, and contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing a comonomer, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.
The solid support can be platinum or palladium, and the method can further comprise the steps of contacting the solid support with a compound comprising a nitrile or isonitrile group and a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface, and contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m in are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.
The solid support can be copper, and the method can further comprise the steps of contacting the solid support with a compound comprising a hydroxamic acid group and a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface, and contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator and disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.
The solid support can be a polymer comprising a reactive functional group, and the method can further comprise the steps of contacting the solid support with a compound comprising a functional group which can react to form a bond with the reactive functional group and a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having immobilized unsaturated aliphatic group, and contacting the unsaturated aliphatic groups of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.
The polymeric solid support can be polystyrene. The reactive functional group of the polymeric solid support can be an amine group or a hydroxyl group and the compound is represented by the following structural formula: 
wherein Y is a leaving group, L is a linking group, and R10 is a substituted or unsubstituted alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl. Y can be: 
wherein R6 and R7 are each, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.
The method can further comprise the step of derivatizing a solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups. The solid support can be doped or undoped silica, alumina, quartz or glass, and the solid support can be derivatized by contacting it with a compound comprising a silane group or a carboxylic acid group and a latent thiol group.
The solid support can be platinum or palladium, and the solid support is derivatized by contacting it with a compound comprising a nitrile or isonitrile group and a latent thiol group.
The solid support can be a polymer comprising reactive functional groups, and the solid support is derivatized by contacting it with a compound comprising a functional group which can react to form a bond with the reactive functional group and a latent thiol group. The polymeric solid support can be polystyrene. The reactive functional group of the polymeric solid support can be an amine or a hydroxyl group and the solid support can be derivatized by contacting the solid support with a compound represented by the following structural formula: 
wherein Y is a leaving group, L is a linking group, and R4 is a thiol protecting group, thereby forming a solid support having immobilized latent thiol groups. Y can be: 
wherein R6 and R7 are each, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group. R4 can be: 
wherein R6 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.
In another embodiment, the invention is directed to a method of making a solid support having immobilized thiol groups, comprising the steps of contacting a glass solid support with a silane compound represented by the following structural formula: 
wherein X is a halogen, and R1, R2 and R3 are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one xcex1,xcex2-unsaturated carbonyl, provided that at least one of R1, R2 or R3 is an alkenyl group or a group having at least one (xcex1,xcex2-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface, contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula: 
wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups, and contacting the latent thiol groups with a disulfide reducing agent, thereby forming a solid support having immobilized thiol groups.
In another embodiment, the invention is directed to a method of forming an array of nucleic acids inmmobilized on a solid support including forming an amine-derivatized region on the support, treating the amine-derivatized region with a thiolating agent such that latent thiol groups immobilized on the support are formed, activating the latent thiol groups, contacting the activated thiol groups with a plurality of nucleic acids comprising acrylamide functional groups, and forming a covalent bond between the two groups, thereby forming an array of nucleic acids immobilized on the solid support. In alternate embodiments, each nucleic acid contained in the array includes a nucleotide sequence identical to or substantially identical to, the nucleotide sequence of the other nucleic acids of the array, or nucleic acids with a plurality of nucleotide sequences are contained in the array. The solid support can include a plurality of amine-derivatized regions. The method can further include a step of blocking any unbonded reactive thiol groups remaining following the binding of the nucleic acids to the thiol groups.
In another aspect, the invention is directed to a kit for attaching nucleic acids to a solid support including a solid support component including a plurality of immobilized latent thiol groups and instructions for activating the thiol groups to form covalent bonds with nucleic acids including acrylamide functional groups. Such kits can also include an activator component, an acrylamide functional nucleic acids component, a blocking component and/or a wash buffer.
In an alternate embodiment, the invention is directed to a kit for attaching nucleic acids to a solid support including a solid support component including a plurality of immobilized latent thiol groups and nucleic acids including acrylamide functional groups. In a particular embodiment, the nucleic acids are immobilized on the solid support by a covalent bond between the immobilized thiol groups and the nucleic acids. Such kits can also include an activator component, a blocking component and/or a wash buffer.
In another aspect, the invention is directed to a method for detecting or separating target nucleic acids from other components contained in a sample including providing a solid support comprising a plurality of immobilized nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid, wherein the nucleic acids are immobilized by a covalent bond formed between a thiol group immobilized on the solid support and an acrylamide functional group contained on the nucleic acid, contacting the immobilized nucleic acid with the test sample, and hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences, thereby separating target nucleic acids from other components contained in the sample. After detection or separation, the target nucleic acids can be amplified. The method can be used in an assay for detecting a contaminant in a sample, for medical diagnosis of a medical condition, for genetic and physical mapping of genomes, for monitoring gene expression and for DNA sequencing.
In another embodiment, the invention is directed to a method for detecting or separating target nucleic acids from other components contained in a sample including providing a solid support comprising a plurality of immobilized thiol groups, contacting the thiol groups with a plurality of nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid and acrylamide functional groups, forming a covalent bond between the two groups, thereby immobilizing the nucleic acids on the solid support, contacting the immobilized nucleic acids with the test sample, and hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences, thereby detecting or separating target nucleic acids from other components contained in the sample. After detection or separation, the target nucleic acids can be amplified. The method can be used in an assay for detecting a contaminant in a sample, for medical diagnosis of a medical condition, for genetic and physical mapping of genomes, for monitoring gene expression and for DNA sequencing.